Abstract B133: Chromatin flow cytometry based quantification of cell type specific alterations in histone methylation states resulting from in vitro and in vivo EZH2 inhibitor treatment

Autor: Alice McDonald, Stephen J. Blakemore, Sarah K. Knutson, Robert A. Copeland, Heike Keilhack, Natalie Warholic, Jesse Smith, Christopher Plescia
Rok vydání: 2015
Předmět:
Zdroj: Molecular Cancer Therapeutics. 14:B133-B133
ISSN: 1538-8514
1535-7163
Popis: Introduction: Evaluating the effects of epigenetic modulators on the methylation state of histones in heterogeneous cell populations has, to this point, been limited to bulk assessment of cell lysates or purified histones. Hence, determining the differential effects of these compounds on individual cell types within a complex population has remained elusive. Methods: In order to monitor the pharmacodynamics of tazemetostat (EPZ-6438), an EZH2 inhibitor that has demonstrated clinical activity in multiple oncology indications, with greater sensitivity and specificity in phase 2 clinical trials and beyond, we have developed a chromatin flow cytometric assay that quantifies trimethylation of lysine 27 of histone H3 (H3K27me3) and total H3 levels while simultaneously allowing for immunophenotyping of discrete populations by surface marker composition in human and murine blood leukocytes. Results: Tazemetostat treatment of human lymphoma cell lines for 4 days resulted in dose-dependent reductions in H3K27me3 levels when quantified both by traditional histone western blotting and chromatin flow cytometry. Furthermore, both assays were able to discriminate between EZH2 mutant lymphoma cell lines which lack H3K27 dimethylation (H3K27me2) and wild-type lines in which the H3K27me2 is present and reduced in a dose responsive manner. To determine if the assay was capable of discriminating methylation states of specific cell populations found in whole blood from an in vivo model, we performed a 7-day study in mice treated with increasing dosages of tazemetostat (EPZ-6438). Quantification of H3K27me3 levels in blood leukocytes revealed differential responses to inhibitor treatment based on cell type. Monocytes and NK cells were found to be highly sensitive to tazemetostat, demonstrating 85% and 70% reduction in H3K27me3 respectively. B-cells were moderately sensitive to tazemetostat treatment, exhibiting a 21% reduction in H3K27me3. Granulocytes and T-cells were the least sensitive with a 7% and 3% reduction in H3K27me3 respectively. Conclusions: These results demonstrate that chromatin flow cytometry is an effective means to monitor cell type specific changes in methylation state upon compound treatment in vivo. Given the advantages of this methodology over conventional measures of histone methylation, chromatin flow cytometry will be used to monitor pharmacodynamics of H3K27me3 inhibition in whole blood leukocytes in the ongoing phase 2 and future clinical trials of tazemetostat. Citation Format: Christopher Plescia, Sarah Knutson, Natalie Warholic, Alice McDonald, Heike Keilhack, Jesse Smith, Robert Copeland, Stephen Blakemore. Chromatin flow cytometry based quantification of cell type specific alterations in histone methylation states resulting from in vitro and in vivo EZH2 inhibitor treatment. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B133.
Databáze: OpenAIRE
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